TWI435951B - Acidic electrolyzed water and composition containing the same - Google Patents

Description

Electrolyzed acidic water and its constituents

The invention relates to an electrolyzed water and a composition thereof, in particular to an electrolytic acidic water and a composition thereof which have antibacterial, anti-inflammatory, anti-oxidation, promote wound healing, inhibit cancer cells but are not cytotoxic and do not cause allergies. Things.

As consumers' health awareness gradually rises, in order to avoid skin damage caused by chemical makeup products, the composition of cosmetic products is subject to more and more strict inspection.

The electrolytic acidic water is electrolyzed by a brine or a dilute hydrochloric acid by an electrolytic bath to produce electrolytic acidic water of a colorless high-concentration hydrogen ion at the anode. Electrolytic acid water is harmless to the environment and easy to mass-produce, so it can be widely used. For example, electrolyzed super-acid water having a pH of less than 2.5 is known to have a bactericidal function, and a medical institution in Japan uses electrolyzed super-acid water as a disinfecting wound and sterilization. Secondly, in agriculture, there is also the use of electrolytic super-acid water instead of pesticides to sterilize, and then alkaline water to improve soil pH. In addition, Japanese manufacturers have also developed the use of electrolytic acid water to clean residual metal ions on the surface of the wafer to reduce the amount of chemicals used.

For sterilization purposes, conventional techniques must use electrolytic super-acid water having a pH of less than 2.5 to have a bactericidal function. However, if the electrolyzed super-acid water with bactericidal function is directly applied to the skin, it may cause damage to the skin cells due to the pH being too acidic. If the electrolyzed super acid water is diluted to a weak acidity, the bactericidal effect is not good.

In view of the above, there is a need to provide an electrolytic acidic water to overcome the above-mentioned doubts of conventional electrolytic acidic water, and to further develop its application to cosmetic compositions.

Therefore, an aspect of the present invention provides an electrolytic acidic water which utilizes electrolytic acidic water having weak acidity, low electrical conductivity and high oxidation potential, which has bacteriostatic, anti-inflammatory, anti-oxidation, and promotes wound healing. It inhibits the effects of cancer cells, but it is not cytotoxic and does not cause allergies.

Another aspect of the present invention provides a composition for disinfecting a skin surface, comprising the above-mentioned electrolytic acidic water as an active ingredient for skin surface disinfection, and a cosmetic composition and a pharmaceutical composition which can be safely applied to skin surface disinfection. Or other composition.

According to the above aspect of the invention, an electrolytic acidic water is proposed. In one embodiment, the electrolytically acidic water is characterized by having a pH of from 3.0 to 4.8, an electrical conductivity of from 150 microseconds (μs/cm) to 300 μs per centimeters, and an oxidation of from 350 millivolts (mV) to 450 mV. The potential, and this electrolytic acidic water system is used to inhibit the growth of Pseudomonas aeruginosa, Staphylococcus aureus, Acne bacillus and Escherichia coli, but is not cytotoxic and does not cause allergies.

According to another aspect of the present invention, a composition for disinfecting a skin surface comprising 1% by weight to 30% by weight of the above-mentioned electrolytic acidic water as an active ingredient for skin surface disinfection to inhibit Pseudomonas aeruginosa, gold Staphylococcus aureus, acne bacillus, and Escherichia coli are grown but not cytotoxic, and the composition has anti-inflammatory effects, anti-oxidation effects, promotes proliferation of mouse fibroblasts, and inhibits proliferation and migration of human skin melanoma cancer cells.

The electrolytic acidic water and the cosmetic composition thereof of the present invention are made of electrolytic acidic water having weak acidity, low electrical conductivity and high oxidation potential, and have the effects of inhibiting bacteria, anti-inflammatory, anti-oxidation and promoting wound healing, but It is not cytotoxic and does not cause allergies. It has been proven to inhibit the proliferation and migration of melanoma cells in the skin, so it can be safely applied to cosmetic compositions, pharmaceutical compositions or other compositions for disinfection of the skin surface.

As described above, the present invention provides an electrolytic acidic water and a cosmetic composition thereof, which utilizes electrolytic acidic water having weak acidity, low electrical conductivity, and high oxidation potential to inhibit Pseudomonas aeruginosa, Staphylococcus aureus, and acne on the skin surface. Growth of bacilli and E. coli but not cytotoxic.

The term "electrolytic acidic water" as used in the present invention refers to electrolytic acidic water which produces a high concentration of hydrogen ions at the anode during the process of utilizing seawater to form a salt by an ion exchange membrane electrodialysis salt making method. Please refer to FIG. 1 , which is a schematic diagram showing a manufacturing process of electrolytic acidic water according to an embodiment of the present invention. First, seawater is introduced into the seawater introduction tank 101, and the seawater is initially filtered by the sand filter bed 103. The seawater after the preliminary filtration is introduced into the ion exchange membrane electrodialysis machine 105 and the evaporation crystallizer 107 to perform ion exchange to separate the raw material water, the crystal salt, and the bitter brine, and the raw material water is sent to the raw water tank 109. The raw material water is filtered by a filter 113, and then introduced into a reverse osmosis water purifier 115 to purify water to obtain clean pure water.

Then, pure water is added to the mixing tank 117. Thereafter, the pure water is introduced into a conventional electrolysis machine 119, and the current intensity of the electrolysis machine 119 is set, and forced electrolysis is performed to generate electrolytic acid water 123 having a high concentration of hydrogen ions at the anode, and the obtained electrolytic acidic water 123 can be obtained. Other applications and feature evaluation.

Further, the electrolytic acidic water is weakly acidic, and its pH value is from pH 3.0 to pH 4.8, so that it does not irritate the skin. Secondly, the electrolytic acidic water has an electrical conductivity of 150 μs/cm to 300 μs/cm and an oxidation potential of 350 millivolts (mV) to 450 mV, and after further analysis, can effectively inhibit Pseudomonas aeruginosa, Staphylococcus aureus on the skin surface, The growth of acne bacteria and E. coli is not cytotoxic.

In an exemplary embodiment, the electrolytic acidic water may have a pH of from 3.1 to 4.5, an electrical conductivity of from 170 μs/cm to 250 μs/cm, and an oxidation potential of from 350 mV to 450 mV. In another example, the electrolytic acidic water may have a pH of from pH 3.3 to 4.2, an electrical conductivity of from 190 μs/cm to 210 μs/cm, and an oxidation potential of from 390 mV to 420 mV.

It is worth mentioning that the acid-base of the electrolytic super-acid water obtained by the prior art needs to reach pH 2.5 to have antibacterial ability. The electrolytic acid water of the invention has weak acidity (pH 3.0 to pH 4.8), low electrical conductivity and high oxidation. Potential, effectively inhibits the growth of Pseudomonas aeruginosa, Staphylococcus aureus, Acne bacillus and Escherichia coli on the skin surface but is not cytotoxic, and has been proven to have anti-inflammatory, anti-oxidant, and wound healing effects, but not cytotoxic It does not cause allergies, and it has been confirmed to inhibit the proliferation and migration of melanoma cells in the skin, so it can be safely applied to cosmetic compositions, pharmaceutical compositions or other compositions for disinfecting the skin surface.

In one example, the composition for skin surface disinfection of the present invention may comprise from 5 weight percent to 20 weight percent of the above-described acidic electrolyzed water as an active ingredient for skin surface disinfection.

The following examples are provided to illustrate the application of the present invention, and are not intended to limit the present invention, and those skilled in the art can make various changes and modifications without departing from the spirit and scope of the invention.

Example 1: Preparation of electrolytic acidic water

In this embodiment, electrolytic salt water having a high concentration of hydrogen ions is generated at the anode during the salt-making process by ion-exchange membrane electrodialysis. In addition, please refer to FIG. 1 , which is a schematic diagram showing a manufacturing process of electrolytic acidic water according to an embodiment of the present invention. First, seawater is introduced into the seawater introduction tank 101, and the seawater is initially filtered by the sand filter bed 103. The initially filtered seawater is introduced into the ion exchange membrane electrodialysis machine 105 and the evaporative crystallizer 107 to perform ion exchange to separate the raw material water, the crystalline salt and the bitter brine, wherein the raw material water is sent to the raw water tank 109, and the bitter brine is stored. In the storage tank 111. The raw material water is filtered by a filter 113, and then introduced into a reverse osmosis water purifier 115 to purify water to obtain clean pure water.

Then, pure water is added to the mixing tank 117. Thereafter, pure water is introduced into an electrolysis machine 119 (for example, OSG Melta Super Alkalizer, model NDX-3000, OSG, Japan), and the current intensity of the electrolysis machine 119 is controlled to perform forced electrolysis to obtain electrolytic acidic ionized water at the anode.

The pH, conductivity and oxidation potential of the above-mentioned electrolytic acidic water can be determined by any commercially available instrument such as a pH meter (for example, NeoMet pH/mV/ORP/Temp Meter, model pH-220L, istek, Inc., Korea). Conductivity meters (e.g., Conductivity Meter, Model 430C, istek, Inc., Korea) or other instruments having the same function, but the measurement of the above values is well known to those of ordinary skill in the art to which the present invention pertains, and no further description is provided. . The above-mentioned electrolytic acidic water has a pH of 3.35 and an electrical conductivity of 199.6 ± 2.1 μs.

Example 2: Evaluation of the composition of electrolytic acidic water

In this embodiment, the electrolytic acidic water of Example 1 is further utilized by a commercially available triple quadrupole mass spectrometry (MS/MS), such as a Finnigan TSQ Quantum Ultra tandem three-stage quadrupole mass spectrometer (Thermo Fisher Scientific Inc. , San Jose, CA, USA), analyzing its composition. In short, firstly, the electrolytic acidic water is injected into the NanoSpray ion source of the mass spectrometer at a flow rate of 1 μL/min using a syringe, and the positive mode is selected, and the ion is performed by using a spray voltage of 1800 V. The mass spectrometry was then carried out with a mass-to-charge ratio (m/z) with a scan range of 20 to 135.

In addition, the electrolytic acid water of Example 1 was analyzed by an induced couple plasma mass spectrometry (ICP-MS) for containing, for example, chromium ions (Cr ⁶⁺ ), mercury ions (Hg ²⁺ ), cadmium ( Heavy metals such as Cd), lead (Pt) and arsenic (As); and methylmercury [methyl mercury; Hg(CH ₃ ) ⁺ ] and dimethyl arsine hydroxide; As(CH ₃ ) ₂ (HO )] and other organic metals; and other metal elements such as magnesium ions (Mg ²⁺ ), calcium ions (Ca ²⁺ ), sodium ions (Na ²⁺ ), potassium ions (K ⁺ ), and the like.

Referring to FIG. 2, there is shown a liquid chromatography mass spectrometry (LCMS) analysis map of electrolytic acidic water according to the first embodiment of the present invention, wherein the horizontal axis is mass-to-charge ratio (m/z), longitudinal The axis is the relative absorption rate (%).

It can be observed from the LCMS analysis chart of Fig. 2 that the electrolytic acidic water of the first embodiment contains potassium ions (K ⁺ ; m/z = 39) and potassium chloride hydrated with protons (KCl + H ⁺ ; m / z = 75) Other minerals, but no heavy metals. Secondly, the electrolytic acidic water of the first embodiment further contains an inhibitory inflammatory factor such as magnesium ion and zinc ion; the magnesium ion is coupled with a chloride ion (Mg ³⁵ Cl ⁺ ; m/z = 59) or with a chloride ion isotope ( ³⁷ Cl Coupling (Mg ³⁷ Cl ⁺ ;m/z=61); magnesium dichloride (Mg ³⁵ Cl ₂ +H ⁺ ;m/z=95) hydrated with a proton, chlorine ( ³⁵ Cl) and chlorine hydrated with a proton Isotope ( ³⁷ Cl) magnesium (Mg ³⁵ Cl ³⁷ Cl+H ⁺ ; m/z = 97), and a dichloroisotope ( ³⁷ Cl) magnesium hydrated with a proton (Mg ³⁷ Cl ₂ +H ⁺ ;m/ z=99). Since chlorine isotope (Cl ³⁷ ) accounts for about 25% in nature, the content of dichloroisotope ( ³⁷ Cl ) magnesium (Mg ³⁷ Cl ₂ +H ⁺ ;m//z=99) is small. Furthermore, the hydration of metal ions with water molecules can also constitute hydrated micelles, such as magnesium dichloride and a proton and hydrated with one or two water molecules [(Mg ³⁵ Cl ₂ +H ₂ O+H ⁺ ;m /z = 113) and (Mg ³⁵ Cl ₂ + 2H ₂ O + H ⁺ ; m / z = 131)]. In addition, the electrolytic acidic water further contains other metal ions, for example, zinc ions are coupled with a chloride ion (Zn ³⁵ Cl ₂ ⁺ ; m/z = 100), and calcium ions are coupled with a hydroxyl group (CaOH ⁺ ; m/z = 57). The above results confirmed that the magnesium-containing ions of the electrolytic acidic water of Example 1 may provide an anti-inflammatory effect, and the zinc-containing ions may provide an antibacterial effect.

Example 3: Evaluation of Cell Safety of Electrolyzed Acidic Water

This example measures the cytotoxicity using a weakly acidic solution similar to that of the electrolytic acid water of the first embodiment.

First, a fibroblast strain (mouse skin fibroblast cell line; BCRC 60008) is cultured in a cell culture medium by a sterile technique, wherein the cell culture medium is Dulbecco's Modified Eagle's Medium (Dulbecco's Modified Eagle's Medium, DMEM; GIBCO Cat. No. 12800-017, Invitrogen Ltd.) and an additional 10% by volume of fetal bovine serum (FBA; SAFC) Cat. No. 12003C, SAFC Biosciences Inc.) and 1 volume percent antibiotic (containing penicillin, streptomycin and antifungal agents, penicillin-streptomycin-fungizone; HyClone Pen/Strep/Fungizon, Cat. No. SV30079.01 Thermo Fisher Scientific Inc.).

The above cell lines were cultured in a 96-well cell culture dish at a cell density of 5 × 10 ⁶ cells/well, and then cultured at 37 ° C and maintained at a humidity of 5% carbon dioxide for at least 24 hours. Thereafter, 10 volume percent to 30 volume percent of the electrolytic acid water of Example 1, the pH acid solution of pH 4.10, and the dilute aqueous hydrochloric acid solution of pH 4.10) or an equal volume of phosphate buffered saline (PBS). , added to the above cells, wherein the PBS system was used as a control group. After 18 hours of culture, the cell growth condition of the cells was observed and the cell survival rate was calculated, and the results are shown in Figs. 3A to 3L and Table 1.

Referring to Figures 3A through 3L, there are shown cell growth scenarios for cytotoxicity assays in accordance with several embodiments of the present invention. Among them, the 3A, 3E, and 3I are the cell growth conditions of the control group, respectively. 3B, 3F, and 3J are cell growth scenarios in which 10% by volume, 20% by volume, and 30% by volume of the electrolytic acidic water of Example 1 were added, respectively. 3C, 3G, and 3K are cell growth scenarios in which 10 volume percent, 20 volume percent, and 30 volume percent of the fruit acid solution were added, respectively. The 3D, 3H, and 3L are cell growth scenarios in which 10 volume percent, 20 volume percent, and 30 volume percent dilute aqueous hydrochloric acid solution were added, respectively. As for the cell viability of the first table, the cell survival rate of the control group was 100%.

From the results of the 3B, 3F, 3J and 1st tables, the cell viability of the electrolytic acidic water of Example 1 after 18 hours of culture can be more than 150% compared with the cells of the control group. The cell survival rate exceeding the control group, which represents the electrolytic acidic water of the first embodiment, can promote the proliferation of the mouse skin fibroblast strain, and indeed achieves the object of the present invention.

However, when the acid solution having a pH value close to that of electrolytic acid water is added to DMEM, not only the medium is changed to yellow in an instant, but after 18 hours of cultivation, the death of the mouse skin fibroblast strain is caused, and the concentration is added. The higher the cell death situation, the more severe, as shown in the results of Figures 3C, 3G and 3K and Table 1.

As for the dilute hydrochloric acid aqueous solution having a pH value close to that of electrolytic acid water, after 10 hours of incubation, 10 to 20% by volume of dilute aqueous hydrochloric acid solution did not significantly kill the mouse skin fibroblast strain, and the cell survival rate was maintained. More than 90%. However, the addition of 30 volume percent of dilute aqueous hydrochloric acid solution still caused death of the mouse skin fibroblast strain, as shown in the results of the 3D, 3H and 3L and Table 1.

Example 4: Evaluation of the antioxidant effect of electrolytic acidic water

This example utilizes the electrolytic acidic water of Example 1 to determine the superoxide removal in the phenazine methosulfate-β-nicotinamide adenine dinucleotide (PMS-NADH) system. The effect of superoxide anion free radical (hereinafter referred to as superoxide).

First, a 240 μM PMS solution, a 1872 μM NADH solution, and a 600 μM nitroblue-tetrazolium (NBT) solution were prepared in 0.1 M phosphate buffer (pH 7.4). Next, 2 to 20% by volume of the electrolytic acidic water of Example 1 (experimental group) or 0.5 mg/mL of gallic acid (as a positive control), with an equal volume of PMS solution, NADH solution, and NBT After the solution is uniformly mixed, the reaction of the following formula (I) to formula (III) is carried out to produce a superoxide anion radical (O ₂ ‧ ^- ), thereby converting the NBT from yellow to blue, and the diformazan ):

NADH+PMS→NAD ⁺ +PMSH (I)

PMSH+2O ₂ →2 O ₂ ‧ ^- +2 H ⁺ +PMS (II)

NBT (yellow) + 2Cl ^- +4 O ₂ ‧ ^- +4H ⁺ → diformazan (blue) + 4O ₂ + 4HCl (III)

After standing at room temperature for 5 minutes, the absorbance at 560 nm (oxazol) was measured with a spectrophotometer. Among them, the lower the absorbance measured at 560 nm, the stronger the ability (or antioxidant capacity) to remove superoxide anion radicals by electrolytic acid water, and the test results are shown in Table 2.

Please refer to Table 2, which shows the results of detecting electrolytic acidic water using the PMS-NADH-NBT system according to an embodiment of the present invention. It can be seen from the results of the second table that the electrolytic acidic water of the first embodiment exhibits a remarkable anion radical scavenging rate compared with the positive control group, and the electrolytic acidic water of the first embodiment has good antioxidant capacity, and indeed achieves the present invention. The purpose.

Example 5: Evaluation of electrolytic acidic water to promote wound healing

This example uses the electrolytic acidic water of Example 1 to determine the migration ability of fibroblasts, and the effect of electrolytic acidic water on wound healing is evaluated because the migration ability of fibroblasts is related to the effect of wound healing.

First, the fibroblast strain (mouse skin fibroblast cell line; BCRC 60008) is cultured in a cell culture solution by a sterile technique, wherein the cell culture fluid is the same as described above, and it is no longer stated here. The above cell line was cultured in a 6-well cell culture dish at a cell density of 1 × 10 ⁷ cells/well, and then cultured for at least 24 hours at a concentration of 5% carbon dioxide maintained at 37 ° C. Thereafter, 5 volume percent of the electrolytic acidic water of Example 1 or 5 volume percent of reverse osmosis water was added to the above cells, and the reverse osmosis water system was used as a control group. After 48 hours to 72 hours of culture, the cell migration of the cells was observed, and the results were as shown in Figs. 4A to 4E.

Referring to Figures 4A through 4E, there are shown cell growth scenarios for cell migration assays in accordance with several embodiments of the present invention. Among them, Figure 4A shows the cell growth situation at the beginning of the cell migration test. Fig. 4B and Fig. 4D are cell growth conditions in which 5 volumes of reverse osmosis water was added for 48 hours or 72 hours, respectively. 4C and 4E are cell growth cases in which 5 parts by volume of the electrolytic acidic water of Example 1 was added for 48 hours or 72 hours.

From the results of the 4C and 4E, it can be seen that compared with the cells of the control group (Fig. 4B and Fig. 4D), the cell migration ability of the electrolytic acidic water of Example 1 after 48 hours or 72 hours of culture is slightly superior. In the control group, the electrolytic acidic water representing the first embodiment can promote wound healing, and indeed achieves the object of the present invention.

Example 6: Evaluation of the antibacterial effect of electrolytic acidic water 1. In vitro antibacterial test (I)

Firstly, e.g. Pseudomonas aeruginosa strains tested (Pseudomonas aeruginosa; ATCC 9027) and Staphylococcus aureus (Staphylococcus aureus; ATCC 6538), were cultured in modified Lee broth (modified Letheen broth; e.g. BD Difco ^TM MLB MLB , Catalog No. 263010, Becton, Dickinson and Company) On a plate agar medium, the number of colonies was counted after incubation at 35 ° C for 48 hours. Acnes (Propionibacterium acne; ATCC 6919) in the culture broth reinforced Clostridium bacteria (Reinforced Clostridial Medium; e.g. Oxoid CM149) agar plates, cultured at 37 [deg.] C after anaerobic conditions for 72 hours and the colonies. The results are shown in Table 3.

Please refer to Table 3, which shows the bacteriostatic effect of detecting electrolytic acidic water according to an embodiment of the present invention. From the results of the third table, it is understood that the electrolytic acidic water obtained in the first embodiment can effectively inhibit the growth of Pseudomonas aeruginosa, Staphylococcus aureus, and Acne bacillus, and the object of the present invention can be achieved.

2. In vitro antibacterial test (II)

In addition, the bacterial strains of the test strains such as Staphylococcus aureus (ATCC 29213) and Escherichia coli (ATCC 35210) are appropriately diluted to a standard concentration of McFarland 0.5 using sterile saline. ×10 ⁸ CFU/mL). Next, after each 1 mL of the bacterial suspension was centrifuged at 9000 rpm for 3 minutes, the supernatant was removed, and the bacteria were treated with 0.95 mL of acidic water (experimental group) or physiological saline (control group) for 30 seconds, After 60 seconds, 180 seconds, 300 seconds or 900 seconds, it was centrifuged for another 2 minutes, rinsed once with sterile physiological saline, and then suspended in sterile physiological saline. Each group of bacterial suspensions was diluted 10 ⁴ times, and 10 μL of each was cultured on a blood agar plate (Columbia agar with 5% sheep blood; Becton, Dickinson and Company, Sparks, MD, US), and cultured at 35 ° C. After the hour, the number of colonies was counted, and the results are shown in Table 4 and Figure 5.

Please refer to Table 4 and Figure 5, which show the results of an in vitro bacteriostatic test according to an embodiment of the present invention, wherein the horizontal axis of Fig. 5 represents the exposure time of the test strain to the electrolytic acidic water of Example 1. ), and the vertical axis of Fig. 5 is the bacterial growth rate (%).

From the results of the fourth table and the fifth graph, it can be seen that Staphylococcus aureus and Escherichia coli are respectively exposed to the electrolytic acidic water of Example 1 for 5 seconds or 15 minutes, and can effectively inhibit 89% of Staphylococcus aureus (Fig. 5 The growth of No. 1) or 52% of Escherichia coli (Fig. 5) can indeed achieve the object of the present invention.

3. Skin application test

In this embodiment, one side of the skin was washed with electrolytic acidic water of Example 1 as an experimental group, and the other side of the skin was washed with reverse osmosis (RO) water as a control group. After the cleaning, the skin samples on both sides were sampled by cotton swabs, and the sampled cotton swabs were placed on an ATP luminometer (3M) to measure the relative absorbance. The results are shown in Table 5.

From the results of the fifth table, it is understood that the electrolytic acidic water of the first embodiment can effectively reduce the number of bacteria on the surface of the skin, and the object of the present invention can be achieved.

Example 7: Evaluation of anti-inflammatory effect of electrolytic acidic water

This example uses human peripheral blood mononuclear cells (PBMCs) to evaluate the anti-inflammatory effect of electrolytic acidic water.

Heparin was used to treat venous blood (h) from adults (25 to 40 years old). After removing the plasma, the buffy coat was treated with a lymphocyte separation solution (Ficoll-Hypaque cushion, specific gravity 1.077 g/mL; Sigma). - Aldrich Co., MO, USA) After forming a clear interface, it was placed on a horizontal centrifuge and centrifuged at 300 times gravitational acceleration (xg) for 20 minutes to form a layer. Next, the PBMCs layer was aspirated, and the cell concentration was adjusted to 1.0 × 10 ⁶ cells/mL with RPMI-1640 medium (Sigma-Aldrich Co., MO, USA). Then, a PBS solution containing gallium chloride (GaCl ₃ ) was added to the PBMCs for cell culture, and the final concentrations of gallium chloride were 0.01 μg/mL, 0.1 μg/mL, 1 μg/mL, 10 μg/mL, and 50 μg/ mL, 100 μg/mL or 1000 μg/mL.

Please refer to FIG. 6A to FIG. 6B , which illustrate the treatment of electrolytic acid water of the first embodiment using physiological saline (control group), 5 volume percent, 10 volume percent or 20 volume percent according to an embodiment of the invention. The concentration (pg/mL) of TNF-α (Fig. 6A) and IL-4 (Fig. 6B) after PBMCs was changed.

From the results of FIGS. 6A to 6B, it was found that the PBMCs treated with the above-mentioned concentration of the electrolytic acidic water were used in comparison with the control group-treated PBMCs without stimulation with lipopolysaccharide (LPS). There is no significant change in the concentration of cytokines (e.g., TNF-[alpha], IL-4) caused by activation of PBMCs.

For similar results, please refer to FIG. 6C to FIG. 6F, which illustrate the electrolysis of the first embodiment using physiological saline (control group), 5 volume percent, 10 volume percent or 20 volume percent according to an embodiment of the present invention. Acidic water, which treats changes in cytokines in four T cells, respectively. Among them, the 6C figure shows the change of the IFN-γ concentration (pg/mL) of the type 1 helper T cell (T helper 1 cell; Th1), and the 6D figure shows the IL-13 concentration of the Th2 cell (pg/mL). Changes, Figure 6E shows changes in TGF-β concentration (pg/mL) of regulatory T cells (Tregs T cells), while Figure 6F shows changes in IL-17A concentration (pg/mL) of Th17 cells. .

From the results of Fig. 6C to Fig. 6F, it can be seen that, compared with the control group treated Th1 cells, Th2 cells, Treg cells, and Th17 cells, the above-mentioned concentration of electrolytic acid water treatment of Example 1 was used without LPS stimulation. The above four T cells also did not cause the above-mentioned T cell activation to cause a significant change in the concentration of cytokines such as TNF-α, IL-4, IFN-γ, IL-13, TGF-β, and IL-17A.

However, treatment of the above cells with endotoxin (e.g., LPS) of Gram-negative bacteria causes a vigorous immune response, resulting in a significant change in the concentration of various cytokines in the above T cells. Please refer to FIG. 7A to FIG. 7B, which illustrate PBMCs according to an embodiment of the present invention, after being stimulated by LPS (0.2 μg/mL), 20% by volume of electrolytic acid water of Example 1 (slanted line length) Article) After treatment of PBMCs or untreated PBMCs (blank strips), TNF-α, IL-4 (Fig. 7A), IFN-γ, IL-13, TGF-β, and IL-17A (Fig. 7B) The concentration (pg/mL) varies.

From the results of Fig. 7A to Fig. 7B, it can be seen that the PBMCs can significantly reduce the PBMCs by treating the cultured PBMCs with 20% by volume of the electrolytic acidic water of Example 1 under the stimulation of lipopolysaccharide (LPS). Changes in the concentration of cytokines such as TNF-α (i.e., activation markers of monocyte/macrophages), IL-4 (Fig. 7A), IFN-γ, IL-13, and IL-17A (Fig. 7B) represent Electrolyzed acidic water does effectively inhibit the activation of monocytes/macrophages associated with inflammatory responses. Secondly, treatment of the above cultured PBMCs with 20% by volume of electrolytic acid water of Example 1 under LPS stimulation also helped to reduce the cytokine IFN-γ (ie, the activation marker of Th1 cells) and IL-13 (ie Th2). The concentration of the cell activation marker) further inhibits the activation of Th1 cells and Th2 cells. In addition, treatment of the above cultured PBMCs with 20% by volume of electrolytic acid water of Example 1 under LPS stimulation significantly reduced the release of IL-17A (i.e., activation marker of Th17 cells) caused by LPS, however, TGF- There was no significant change in β (ie, the activation signature of Treg cells).

In summary, treatment of the above PBMCs with the electrolytic acidic water of Example 1 without stimulation by LPS did not cause activation of PBMCs and caused a significant change in the concentration of cytokines. However, under the stimulation of LPS, it is known that the concentration of TNF-α (i.e., the activation marker of monocyte/macrophage) is reduced, and the treatment of the above PBMCs by the electrolytic acidic water of Example 1 can effectively inhibit the inflammatory reaction. Activation of related immune cells. Secondly, it can be known from the decrease of the concentration of IL-13 (i.e., the activation marker of Th2 cells) that the treatment of the PBMCs by the electrolytic acidic water of the first embodiment can also help inhibit the activation of immune cells associated with certain allergic reactions. .

Example 8: Evaluation of non-sensitization of electrolytic acidic water

This example further evaluated whether the electrolytic acidic water of Example 1 caused an allergic reaction by using mast cells.

Mast cells can secrete a variety of inflammatory mediators, such as granules of mast cells containing beta-hexosaminidase. When mast cells are activated immunologically, for example, via interaction with FcεRIs, β-hexosaminidase is usually released by exocytosis. Stimulation caused by cross-linking of antigens can activate mast cells or basophils and de-granulation of these two types of cells to release β-aminohexose Enzyme. By detecting changes in the concentration of β-aminohexosidase, it can be applied to assess the sensitization of natural or synthetic substances. In view of the fact that rat basophilic leukemia cells (RBL-2H3 cells) can mimic the multiple functions of mucosal mast cells, this example uses this cell model to evaluate whether the electrolytic acidic water of Example 1 causes an allergic reaction. .

For the evaluation of the degranulation of RBL-2H3 cells by antigen, reference may be made to the β-hexosaminidase activity assay commonly used in the prior art. Briefly, first, RBL-2H3 cells (ATCC CRL-2256) were implanted into a 75 cm ² culture dish and contained Dubecco's Modified Eagle Medium; DMEM; Gibco, Grand Island , NY, USA, in which 10% by volume of fetal bovine serum (FBS) was added, and 100 U/mL of penicillin (penicillin) and 100 g/mL of streptomycin were added and placed. The cells were cultured in a 37 ° C incubator with 5% CO ₂ , and cell passage was performed at a ratio of 1:2 every 4 days. On the day of the experiment, the cell line was pre-washed and replaced with fresh medium.

Then, RBL-2H3 cells (1 × 10 ⁵ cells/well) were seeded in a 96-well microplate, and cultured at 37 ° C for 4 hours to 6 hours to completely attach the cells to the culture plate. Next, different final concentrations (0 to 20% by volume) of the electrolytic acidic water of Example 1 were added to the cell culture medium, and cultured at 37 ° C for 24 hours, wherein the control group cells were not treated with electrolytic acidic water.

Then, mouse IgE (mouse anti-DNP IgE; mIgE-DNP) monoclonal antibody against dinitrophenol (DNP) was added to a 96-well microplate, and sensitization was performed at 37 ° C for 16 hours. Groups of cells were not sensitized by IgE. Thereafter, pre-heated Tyre's buffer (Tyrode's buffer; containing 135 mM NaCl, 5 mM KCl, 1.8 mM CaCl ₂ , 1.0 mM MgCl ₂ , 5.6 mM glucose, 20 mM HEPES, and 1 mg/ml BSA; pH 7.4) was used. After washing the IgE-sensitized RBL-2H3 cells in a 96-well microplate twice, then adding a cross-bound antigen, such as dinitrophenol-conjugated bovine serum albumin diluted with a Tie's buffer solution (dinitrophenol- Bovine serum albumin; DNP-BSA; 1 μg/ml), reacted at 37 ° C for 1 hour. Subsequently, the 96-well microplate was placed on an ice bath for 15 minutes to terminate the reaction.

In addition, cells from the positive control group (treated without electrolytic acidic water) were pre-sensitized with IgE and reacted with DNP-BSA antigen after being treated with 10 nM glucosamine steroid (Dexa).

Then, cells in the control group of cells I (treated without electrolytic acidic water, not sensitized with IgE and not reacted with DNP-BSA antigen) were not dissolved. Control group of cells II (treated without electrolytic acidic water but sensitized by IgE and reacted with DNP-BSA antigen), sample group cells (treated by electrolytic acidic water, sensitized by IgE and reacted with DNP-BSA antigen The cells of the positive control group (hereinafter referred to as the Dexa group) were lysed with a 1% Triton X-100 solution.

Thereafter, the supernatant of each group of cells was collected and transferred to another 96-well microplate, and an equal volume of 1 μM p-nitrophenyl-N-acetamido-β-D-glucoside (p-nitrophenyl-N) was added to each well. -acetyl-β-D-glucosaminide; p-NAG; formulated in 0.1 M citrate buffer solution, pH 4.5) as a substrate, and reacted at 37 ° C for 1 hour. Then, 150 μL of the stop solution (0.1 M Na ₂ /NaHCO ₃ , pH 10.0) was added to each well, and the absorption value of the reaction solution of each well at a wavelength of 405 nm was measured using a commercially available microplate reader, and Formula (IV) calculates the inhibition rate (%) of β-aminohexosaminidase of RBL-2H3 cells, and the results are shown in Fig. 8:

Wherein, the definitions of the variables of the formula (IV) are as shown in the sixth table:

The value obtained by the above formula (IV) is converted by the following formula (V), which is the relative release rate (%) of β-aminohexosidase:

Relative release rate of β-aminohexosidase (%) = inhibition ratio of β-aminohexosidase (%) (V)

Please refer to FIG. 8 , which is a bar graph showing the non-sensitization phenomenon of electrolytic acidic water for RBL-2H3 cells according to the first embodiment of the present invention, wherein the horizontal axis represents the concentration of electrolytic acidic water of Example 1 (volume percentage). ;%), and the vertical axis is the relative release rate (%) of β-aminohexosidase.

From the results of Fig. 8, it can be seen that the cells of the positive control group (i.e., the Dexa group) can inhibit the relative release rate of β-aminohexosaminidase in Rg-2H3 cells sensitized by IgE, representing Rg-2H3 cells via IgE. Sensitization and reaction with DNP-BSA antigen does cause an allergic reaction. However, compared with the cells of Control Group II, the sample group treated with the electrolytic acidic water of Example 1 did not increase the relative release rate of β-aminohexocaminase, and the electrolytic acid water representing Example 1 did not cause allergies. .

Example 9: Evaluation of electrolytic acidic water against melanoma hyperplasia and migration

There is currently no literature reported that conductive acid water can be used to fight cancer cells. This example further evaluated the effect of the electrolytic acidic water of Example 1 against cancer cells using human melanoma cell line A375.S2 cells (ATCC CRL-1872).

First, A375.S2 cells were implanted in culture dishes and contained DMEM [Gibco, Grand Island, NY, USA, where 10% by volume of FBS was added, and 100 U/mL of penicillin, 100 g/mL of streptomycin, 0.03% of glutamine and 1 mM of sodium pyruvate were cultured in a 37 ° C incubator at 5% CO ₂ .

Then, A375.S2 cells (1 × 10 ⁵ cells/well) were seeded in a 12-well microplate, and cultured at 37 ° C for 4 hours to 6 hours to completely attach the cells to the culture plate. Next, different final concentrations (0 to 50% by volume) of the electrolytic acidic water of Example 1 were added or not added to the (control group) cell culture solution, and a width of about 200 μL of the micropipette tip was drawn on the culture plate. 1 mm blank scoring, after incubation at 37 ° C for 24 hours, calculate the relative cell survival rate of A375.S2 cells at 0 hours and 20 hours, and use, for example, TScratch software (website: http://www.cse-lab.ethz. Ch/index.php?&option=com_content&view=article&id=363 ) Automated analysis of A375.S2 cells at 0 and 20 hours of cell migration, the results are shown in Figures 9A and 9B, respectively.

Please refer to FIG. 9A and FIG. 9B for showing the relative survival rate of cells for inhibiting proliferation (Fig. 9A) and migration (Fig. 9B) of electrolytic A375.S2 cells according to Example 1 of the present invention (%). The results, wherein the horizontal axis represents the concentration (volume percentage; %) of the electrolytic acidic water of Example 1, and the vertical axis represents the cell relative survival rate (%) of the electrolytic acidic water of the A375.S2 cell in Example 1.

As can be seen from the results of Fig. 9A, as the concentration of the electrolytic acidic water of Example 1 is higher, it is indeed more effective to inhibit the proliferation of A375.S2 cells. Next, as can be seen from the results of Fig. 9B, as the concentration of the electrolytic acidic water of Example 1 is higher, it is indeed more effective to inhibit the migration of A375.S2 cells.

The data obtained in the above experimental examples were expressed as mean ± standard error of mean, and the difference between the groups was analyzed by Student's t-test ( p < 0.05).

In summary, the present invention utilizes electrolytic acidic water having weak acidity, low electrical conductivity and high oxidation potential, and has antibacterial, anti-inflammatory, anti-oxidation, and wound healing effects, but is not cytotoxic and does not cause allergy, and It has been shown to inhibit the proliferation and migration of melanoma cells in the skin. However, it should be added that the present invention describes the electrolytic acidic water of the present invention by a specific process, a specific analysis method, a specific test, a specific test condition, a specific test cell, or a specific device, etc., but the present invention belongs to It is to be understood by those skilled in the art that the present invention is not limited thereto, and other processes, other analytical methods, other tests, and other reaction conditions may be used for the electrolytic acidic water of the present invention without departing from the spirit and scope of the present invention. , other test cells or other equipment.

Next, the electrolytic acidic water of the present invention is confirmed to have the above multiple effects, and thus can be applied to a cosmetic composition, a pharmaceutical composition, a food composition, a cleaning composition or other composition.

It can be seen from the above embodiments of the present invention that the electrolytic acidic water and the cosmetic composition thereof of the present invention have the advantages of utilizing electrolytic acidic water having weak acidity, low electrical conductivity and high oxidation potential, and indeed have antibacterial, anti-inflammatory, anti-oxidation, It promotes wound healing and other effects, but it is not cytotoxic and does not cause allergies. It has been proven to inhibit the proliferation and migration of melanoma cells in the skin, so it can be safely applied to cosmetic compositions, pharmaceutical compositions or other constituents for skin surface disinfection. in.

The present invention has been disclosed in the above embodiments, and is not intended to limit the present invention. Any one of ordinary skill in the art to which the present invention pertains can make various changes without departing from the spirit and scope of the invention. The scope of protection of the present invention is therefore defined by the scope of the appended claims.

101. . . Seawater introduction pool

103. . . Sand filter bed

105. . . Ion exchange membrane electrodialysis machine

107. . . Evaporation crystallizer

109. . . Original sink

113. . . filter

115. . . Reverse osmosis water purifier

117. . . Mixing tank

119. . . Electrolytic machine

123. . . Electrolyzed acidic water

The above and other objects, features, advantages and embodiments of the present invention will become more apparent and understood.

1 is a schematic view showing a manufacturing process of electrolytic acid water according to an embodiment of the present invention.

Fig. 2 is a view showing a liquid chromatography mass spectrometry (LCMS) analysis pattern of electrolytic acidic water according to Example 1 of the present invention.

Figures 3A through 3L show cell growth scenarios for cytotoxicity assays in accordance with several embodiments of the invention.

Figures 4A through 4E show cell growth scenarios of cell migration assays in accordance with several embodiments of the present invention.

Figure 5 is a graph showing the results of an in vitro bacteriostatic test according to an embodiment of the present invention.

6A to 6B are diagrams showing TNF-α (Fig. 6A) and IL after treatment of PBMCs with physiological saline (control group) or different concentrations of electrolytic acid water according to an embodiment of the present invention. -4 (Fig. 6B) concentration change.

6C to 6F are diagrams showing changes in cytokines of four T cells treated with physiological saline (control group) or different concentrations of electrolytic acid water of Example 1 according to an embodiment of the present invention.

7A to 7B are diagrams showing the PBMCs treated with the acidified acidic water (hatched strip) of the first embodiment of the PBMCs according to an embodiment of the present invention, respectively, after treating the PBMCs or the untreated PBMCs (blank strips). Concentration changes of TNF-α, IL-4 (Fig. 6A), IFN-γ, IL-13, TGF-β, and IL-17A (Fig. 6B).

Fig. 8 is a bar graph showing the non-sensitization phenomenon of electrolytic acidic water against RBL-2H3 cells according to Example 1 of the present invention.

Fig. 9A and Fig. 9B show the results of electrolyzed acidic water for inhibiting proliferation (Fig. 9A) and migration (Fig. 9B) of A375.S2 cells according to Example 1 of the present invention.

Claims (9)

An electrolytic acidic water characterized in that the electrolytic acidic water has a pH of from 3.1 to 4.5, an electrical conductivity of from 170 microseconds (μs/cm) to 250 μs/cm, and an oxidation of from 350 millivolts (mV) to 450 mV. The potential is free of silver, and the electrolytic acidic water system is used to inhibit the growth of Pseudomonas aeruginosa, Staphylococcus aureus, Acne bacillus and Escherichia coli but is not cytotoxic and does not cause allergy. The electrolytic acidic water according to the above aspect of the invention, wherein the electrolytic acidic water has a pH of from pH 3.3 to 4.2, an electrical conductivity of from 190 μs/cm to 210 μs/cm, and an oxidation potential of from 380 mV to 420 mV. The electrolytic acidic water according to claim 1, wherein the electrolytic acidic water is used to promote proliferation of mouse fibroblasts. The electrolytic acidic water according to the first aspect of the invention, wherein the electrolytic acidic water system has an anti-inflammatory effect. The electrolytic acidic water according to claim 1, wherein the electrolytic acidic water is used for inhibiting proliferation and migration of human melanoma cells. A composition for disinfecting a skin surface, comprising from 1% by weight to 30% by weight, as in any of Items 1 to 3 of the patent application scope The electrolytic acidic water described in the item is an active ingredient for disinfecting the skin surface to inhibit the growth of Pseudomonas aeruginosa, Staphylococcus aureus, Acne bacillus and Escherichia coli but is not cytotoxic and does not cause allergy, and the composition has anti-inflammatory effect. Promote the proliferation of fibroblasts and inhibit the proliferation and migration of melanoma cells in the skin. The composition for skin surface disinfection according to claim 6, wherein the electrolytic acidic water is from 5 to 20% by weight. A composition for disinfecting a skin surface according to the invention of claim 6, wherein the composition is a cosmetic composition or a pharmaceutical composition or a cleaning composition. The composition for disinfecting a skin surface according to claim 6, wherein the fibroblast is a mouse fibroblast, and the cutaneous melanoma cancer cell is a human skin melanoma cancer cell.